There is significant evidence linking mitochondria to biological aging, with a recent theory proposing that giant mitochondria are involved in the aging of post-mitotic tissues. In these tissues, giant mitochondria appear to evade autophagocytosis, accumulate, and replace normal mitochondria. Techniques to characterize giant mitochondria in the presence of, or after isolation from, normal mitochondria are greatly needed. The short- term goal of this proposal is to characterize and isolate giant mitochondria and establish whether or not these mitochondria affect muscle function. The long-term goal is to use and make available novel technological approaches for uncovering the changes that, at the subcellular and molecular level, define biological human aging. The proposed work utilizes mitochondria from a myoblast model and muscle from Fischer 344 rats. We have four aims: (1) Characterize giant mitochondria in vivo in cell models based on their cell biology. (2) Purify giant mitochondria isolated from these in vivo cell models and then characterize these giant mitochondria biochemically. (3) Compare the proteomes of giant and normal mitochondria isolated from these models. (4) Characterize giant mitochondria in aging skeletal muscle. The first aim uses confocal microscopy, or this technique combined with single cell capillary electrophoresis, to investigate size, DMA content and synthesis, DNA mutations, carbonyl accumulation, ROS production, membrane potential, mitochondrial fusion, and autophagocytosis. The second aim describes the methods to isolate subpopulations of giant and normal mitochondria using free-flow electrophoresis and then characterize these isolated mitochondria using methods suitable for isolated organelles. The third aim utilizes stable isotope labeling in cell cultures to compare the proteomes of giant and normal mitochondria. The last aim investigates age-related changes in muscle containing giant mitochondria. Significance to Health. Giant mitochondria remain poorly characterized, their proteomic and genomic characteristics have not been defined, and their relationship to muscle function has not been investigated. The characterization of this class of mitochondria is highly relevant to the public health not only because of their possible role in aging tissues, but also because of their appearance as a consequence of alcoholism, human toxicity, and multiple diseases.